Evolving spike mutations in SARS-CoV-2 Omicron variants facilitate evasion from breakthrough infection-acquired antibodies

The Chi-Square statistic was used for categorical variables. Continuous variables were compared by using a two-sided Mann-Whitney U-test. Group (1) and (2) were involved in antigenic analyses of the


Monoclonal antibodies
Monoclonal antibodies tested in this study were constructed and produced at Xiamen University. For each antibody, variable genes were codon optimized for human cell expression and synthesized by General Biol (Anhui, China) into plasmids (EIRBdMie) that dual-promoter vector containing constant regions of human IgG1 heavy and light chains.

Construction and production of variant pseudoviruses
Lentiviral-based pseudotyping particle (LVpp) bearing SARS-CoV-2 spikes were produced as previously described 1

Pseudovirus neutralization assays
Neutralization assays were performed by incubating pseudoviruses with serial dilutions of monoclonal antibodies or plasmas and scored by the reduction in cellular fluorescent images. In brief, serially-diluted samples (3-fold series dilutions, from 1:30 to 1:21,870, 1:10 dilution will be supplemented when 1:30 dilution does not show significant inhibitory activity) were pre-incubated with the pseudovirus inoculum (1,800 GFU/well) for 1 hour.
The mixtures were further incubated with the huACE2-H1299 cells pre-seeded in 96-well cell culture plates at 37°C in a CO2 incubator. After a 2-day culture, cellular fluorescent images were acquired by using Opera Phenix's high-content imaging system (PerkinElmer). For each well, the total (H2B-mRuby3-activated) and LVpp-infected (mNeonGreen-activated) cell numbers were determined by the Columbus Image analysis system (PerkinElmer). After normalization with the total cell number, the infection inhibition ratio of each sample at different dilutions was calculated by comparing it with the LVpponly control. IC50 was defined as the dilution at which the relative light units were reduced by 50% compared with the virus control wells (virus + cells) after subtraction of the background in the control groups with cells only. The IC50 values were calculated using nonlinear regression in GraphPad Prism (version 9.3.1). An ID50≥20 was defined as the cutoff value to determine SARS-CoV-2 nAb seropositivity. And an ID50<5 was assigned a value of 5. The performance of the neutralization assay used in our study has been well documented in our previous study 1 .

Recombinant Protein production
Expression plasmids of recombinant spike proteins were constructed encoding for human codon-optimized sequences from wild-type SARS-CoV-2 (MN908947), BA.2 (EPI_ISL_8253179), and BA.4/5 (EPI_ISL_11542465). The ectodomain of these spike fragments (aa 1-1207, furin cleavage site mutated and HexaPro stabilized, fusing with a T4 trimeric motif) of Omicron were customs synthesized by General Biol (Anhui, China) and cloned into EIRBsMie vector, respectively, as previously described 4 . Expression constructs (StriFKHP, StriFK364HP, and StriFKA75HP) were verified by Sanger sequencing after plasmid isolation using the QIAGEN Miniprep kit (QIAGEN). Plasmids encoding proteins were transiently expressed in ExpiCHO cells and purified with a 5 mL Ni sepharose 6 Fast Flow column (Cytiva). Harvested and concentrated proteins are coated in polystyrene or chemiluminescent microplates for subsequent detection.

Anti-spike IgG measurements
Microplates pre-coated with recombinant spike proteins were as previously described 2 .
For detections, serially-diluted samples (5-fold series dilutions, from 1:10 to 1:6250, 100 μl per well) were added to the wells, and the plates were incubated at 37°C for 30 min, followed by washing with PBST buffer [20 mM PBS (pH7.4), 150 mM NaCl, and 0.05% Tween-20]. Then, anti-human IgG (Thermo Fisher Scientific) for 30-min incubation, and then washing with PBST buffer again. Tetramethylbenzidine (Wantai) chromogen solution (100 μl per well) was added to each well. Ten minutes later, the chromogen reaction was stopped by adding 50 μl of 2 M H2SO4, and optical density (OD) 450 nm-630 nm was measured. The IgG titer was defined as the dilution limit to achieve a positive result (greater than the mean plus 3 SDs of ODs of negative controls). Each plate contained three tests of negative control plasmas, and their ODs were used to determine the cutoff value.
Representative data from technical replicates were performed at least twice for plotting.

Prevalence data of subvariants and mutations
The current snapshot of Omicron subvariants data was taken from the EpiCoV database of GISAID

Statistical analysis
Propensity score matching is utilized for matching between different groups. And the age, gender, co-existing medical conditions, illness severity, and sampling days are employed for matching. When there are significant differences in diagnoses and sampling days between groups, priority is given to ensuring matching the first three characteristics. The Friedman test with Dunn's correction was applied to analyze differences among groups.
The Spearman rank correlation coefficient was used for linear correlation analysis between the antibody titers and titers and the R 2 . The prevalence of mutations was fitted by using the exponential growth model. Statistical differences were considered to be significant for two-tailed P values of < 0.05. Statistical analyses were conducted by GraphPad Prism (version 9.3.1) or R software (version 4.1.2).  .0001. ns, not significant. nAb, neutralizing antibody. ID50, half-maximal inhibitory dilution. COI, cutoff index. Data were plotted as the geometric mean with 95% confidence intervals (CI). The percentage in nAb titers plots indicated the percentage of samples above the detection limit.

Supplementary Fig. S2 | Comparisons of nAb and S-IgG titers against spike variants between HCPs from 2-or 3-dose vaccinated individuals with BA.2 breakthrough infections.
Neutralization ( The data were plotted as the mean value of ≥3 technical replicates.  The Chi-Square statistic was used for categorical variables. Continuous variables were compared by using a two-sided Mann-Whitney U-test. Group (1) and (2) were involved in antigenic analyses of the BA.4/5-RBD mutants. yr, years. IQR, interquartile range.